Plants produce diverse bioactive specialized metabolites as part of their defense mechanisms. Frequently, these metabolites are glucosylated and require activation by β-glucosidases (BGLUs). Brassicaceae plants produce thio-glucosides referred to as glucosinolates that have co-evolved with thio-BGLUs known as myrosinases. During immune response in Arabidopsis thaliana, PEN2/BGLU26 myrosinase hydrolyzes indol-3-ylmethyl glucosinolate (I3G) and 4-methoxy-I3G (4MI3G). This hydrolysis leads to production of indol-3-ylmethyl amine (I3A) and 4-O-glucoside of indol-3-yl formamide (4GlcI3F). Related with PEN2 AtBGLU18-32 have been suggested to act as myrosinases based on the presence of conserved substrate recognition R/K residues. To reveal if these putative myrosinases can replace PEN2 in pathogen-triggered IG metabolism, we expressed AtBGLU18, AtBGLU27 and homologus brassinin-associated BGLU (BrBABG) from Brassica rapa fused in frame with the sequence encoding PEN2-specific C-terminal tail, essential to anchor this protein in mitochondrial membrane, under the control of PEN2 promoter in pen2 mutant. AtBGLU27 and BrBABG exhibited activity towards I3G in planta, as confirmed by I3A accumulation in transgenic A. thaliana lines. Comparing to BrBABG, AtBGLU27 showed higher activity towards 4MI3G as shown by elevated 4GlcI3F accumulation. AtBGLU18 was not detected in the investigated transgenic plants. This could be because of the presence of post-translational modifications, conserved in the vast majority of BGLUs, but not in PEN2, AtBGLU27 and BrBABG. This suggests that PEN2 and its closest homologs lost post-translational modifications to function in pathogen-triggered IG metabolism.
